Common to all DNA polymerases is the 5'-3' polymerase activity responsible for incorporation of nucleotides into the elongating strand during DNA replication. In addition to this activity, many polymerases display auxiliary functions such as proofreading and removal of RNA primers, often located in spatially separate domains. The tight coordination of these functions is essential to error-free replication of DNA; defects in thi process result in mutations or other deleterious effects which can lead to cancer and other devastating diseases. While much is known about the average spatial organization and catalytic mechanisms of these functional domains, little is known about how the different functionalities of polymerase work together to ensure proper enzymatic function during DNA replication. The research proposed here utilizes single-molecule fluorescence experiments to probe the discrete conformational states of DNA polymerase I (Pol I) during replication. Knowledge of the relative populations of these states and the likelihood of transitions among them will lead to a better understanding of how a Pol I coordinates its different activities to ensure proper function, and why this process sometimes fails leading to deleterious outcomes.